The human kinome comprises more than 500 kinases (Manning et al. Science, 298, 1912-1934, 2002). Kinases are involved in numerous cell signaling pathways and regulatory mechanisms and play important roles in a wide range of physiological processes and pathological conditions. Consequently, kinases are of considerable scientific and medical interest and represent an important class of drug targets. A number of kinase inhibitors have been approved for treatment of various types of cancer, and many more are currently in various stages of preclinical and clinical development. Imatinib (Gleeve®), the first small molecule kinase inhibitor to be approved for use in humans, is a mainstay of therapy for patients with chronic myeloid leukaemia. (CML). CML arises as a result of a chromosomal translocation in which part of the BCR gene from chromosome 22 is fused to the ABL gene on chromosome 9, producing a BCR-ABL fusion gene. ABL is a tyrosine kinase, and the BCR-ABL fusion results in dysregulation of the ABL tyrosine kinase domain, rendering the protein oncogenic. By inhibiting the kinase activity of the BCR-ABL protein, imatinib can induce a complete remission in most patients with CML.
Most kinase inhibitors target the kinase ATP-binding site, resulting in potential cross-reactivity. Cross-reactivity can sometimes be advantageous, as it offers the ability to multiple kinases using a single agent. However, use of inhibitors that inhibit multiple kinases can complicate the interpretation of experiments, especially if not all of the targets of the inhibitor are known. From a therapeutic standpoint, cross-reactivity may be beneficial, e.g., in situations in which multiple kinases contribute to a disease. However, cross-reactivity may result in undesirable side effects. Significant effort has been devoted to investigating kinase inhibitor specificity. For example, in vitro kinase assays involving recombinantly produced kinases or kinase domains and test substrates (often peptides containing a potential site for phosphorylation), are widely used. Similarly, screens to identify inhibitors of a particular kinase frequently entail assessing the ability of test compounds to inhibit phosphorylation of a test substrate in vitro. While these approaches are conceptually straightforward, they have a number of significant limitations. For example, it has been difficult to express and purify a number of kinases in full length form. Furthermore, in vitro assays inevitably fail to fully recapitulate the native cellular environment.
There is a need in the art for new methods of assessing kinase inhibitor specificity. There is also a need in the art for new methods of identifying kinase inhibitors.